The long-term objective of this project is to better understand the molecular mechanisms that regulate retinal pigment epithelium (RPE)-specific gene expression. The RPE is critical for the function and survival of photoreceptors. Mutations in several RPE-specific or preferential genes cause inherited human retinal dystrophies. RPE abnormalities have also been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness in elderly Americans. However, despite the importance of the RPE, relatively few studies have focused on the regulation of RPE gene expression. That led us to study such mechanisms using human VMD2, the gene responsible for vitelliform macular dystrophy (Best disease), as a model system. Our studies defined the promoter region sufficient to direct RPE-specific expression in transgenic mice and identified microphthalmia-associated transcription factor (MITF) as a potential positive regulator. The goal of this proposal is to extend these findings. First, to define the role of MITF in VMD2 regulation, expression and functional activities of different MITF isoforms will be examined by quantitative PCR and cell transfection assays. Binding of MITF to the bovine Vmd2 promoter in vivo will be analyzed by chromatin immunoprecipitation, Vmd2 expression in Mitf mutant mice will be examined by quantitative PCR, and the activity of the VMD2 promoter in Mitf mutant RPE will be measured using in vivo electroporation. Then, the role of TFE3 and TFEB, two MITF family members, will be similarly examined using cell transfection and gel shift assays. Finally, transgenic mouse studies with VMD2 promoter-lacZ constructs will narrow down the regulatory regions that are necessary for RPE-specific expression, and factors that bind to them will be cloned using the yeast one-hybrid system. Understanding of the detailed mechanisms of VMD2 expression should provide new insights into the regulatory networks for gene expression in the RPE as well as diseases involving abnormalities of RPE gene expression.